7. PROJECT SUMMARY Myocardial infarction resulting from ischemic injury is a prominent and common feature of cardiovascular morbidity and mortality. Cardiac myosin binding protein-C (cMyBP-C) is, by its degradation during proteolysis, an important determinant of myocardial contractile pathogenesis during I-R injury. Briefly, cMyBP-C is a thick filament-associated protein that stabilizes myosin, an important component of the contractile machinery, to regulate sarcomeric structure and function in the heart. Mutations in the cMyBP-C gene account for ~34% of all cardiomyopathy cases, 70% of which are predicted to produce unstable truncated proteins. During I-R injury, we demonstrated that extensive fragmentation of cMyBP-C correlates with altered sarcomeric structure and contractile dysfunction. Therefore, while the short-term goal is to elucidate the proteolytic and pathogenic properties of cMyBP-C in the clinical context of cardioprotection during ischemia-reperfusion (I-R) injury, the long-term goal is to determine the mechanisms by which cMyBP-C stabilizes sarcomeric structure and function in order to confer cardioprotection during I-R injury. More specifically, our preliminary studies show that calpains degrade cMyBP-C into several fragments and that the 29-kDa fragment is the predominant fragment in vitro. Such proteolysis leads to the release of the 29-kDa fragment into the blood stream during I-R injury in mice. Moreover, mass spectrometry analyses confirm that the release of the 29-kDa fragment is associated with the calpain-targeted site (CTS), which is a conserved phosphorylation motif that possibly regulates its cleavage. From a therapeutic perspective, these findings indicate that the ablation of the CTS could result in resistance to calpain-mediated proteolysis, thus abrogating release of the 29-kDa fragment. Therefore, we propose that inhibition of CTS cleavage would secure the structural integrity of cMyBP-C, thus preserving contractile structure and function. However, the clinical and pathogenic significance of cMyBP-C degradation, as well as the properties of its proteolysis, have not been determined and therefore represent a clinically important area of translational research. The goal, therefore, is to determine the correlation between the release of the 29- kDa fragment in the blood and contractile dysfunction, demonstrate its toxic effects in cardiomyocytes and examine how the inhibition of CTS cleavage in cMyBP-C protects the heart from I-R injury. Overall, the proposed research aims to define the stability and function of cMyBP-C in the context of supportive therapy during I-R injury, in general, and heart muscle contractility, specifically. To achieve our goals, Specific Aim 1 will determine the levels of 29-kDa fragment in the blood, according to infarct size and contractile function during I-R injury. Specific Aim 2 will determine the pathogenic properties of the 29-kDa fragment in the context of myosin function. Specific Aim 3 will determine whether site-specific inhibition of the CTS, as defined above, can preserve cMyBP-C stability and function during I-R injury and thus confer cardioprotection. Importantly, once the kinetics of the 29-kDa fragment have validated that this peptide is quantifiable in the serum of wild-type non-transgenic mice with induced I-R injury, we can confirm its potential as a clinically useful readout of post-ischemic myocardial infarction. Our experimental approach is comprehensive, ranging from the analysis of molecular interactions to functional assessments of sarcomeric arrangement and function, both in vitro and in vivo. I-R injury will be induced in wild-type non-transgenic mice to define the sequential release of the 29-kDa fragment and its blood serum levels in relation to infarct size, calpain activities, and myocardial function, compared with controls. Adult mouse cardiomyocytes have been chosen as the model system to investigate the pathogenic properties of the 29-kDa fragment by using recombinant adenoviruses and peptides. To determine the association between the CTS in cMyBP-C and cardioprotection, we will use transgenic mice expressing cMyBP-C in which the CTS has been ablated and bred into the cMyBP-C null background, compared with transgenic mice expressing phospho-mimetic and wild-type cMyBP-C controls. Endpoint measurements include the amount of the 29-kDa fragment in the blood correlated with infarct area and cardiac function, calpain activity, cMyBP-C phosphorylation levels, intracellular Ca2+ transients, Mg2+-ATPase activity, myofilament Ca2+ sensitivity, molecular binding studies, sarcomere structure and function.